In the blood-testing field, assays of interest frequently are limited to tests applied to serum or plasma, rather than to whole blood. The reason is that the cell fraction of whole blood has constituents that may interfere with or obscure the assay. For such assays, a preliminary step is phase separation, in which the cellular fraction is separated and removed.
Whatever the mode of separation that is used, and centrifugation is one of the most common, some means is usually employed to maintain the phase separation after it is achieved, to permit physical separation of the phases without resuspension. One of the most common is the use of a gel having a specific gravity between that of the two phases, as is illustrated by U.S. Pat. No. 4,050,451. Although such a gel is often adequate, it does have weaknesses. One is that it provides only a mechanical barrier, and such barriers can fail. For example, severe agitation of the container in which the gel barrier is in place, can produce failure.
Therefore, prior to the invention there has been a need for a substance to maintain phase separation, that is not simply a breachable wall between the two phases. More specifically, there has been a need for such a substance that provides a substantially irreversible phase separation even in the face of substantial agitation.
Polymeric "coagulants" have been known heretofore, for example, those described in Japanese Kokai No. 58/49410. However, the substances specifically described are for sewage separation, and are polymers in which the pendant amines are fully substituted to create quaternary amines. Blood requires much greater care in handling than sewage so that what works in sewage will not necessarily work in blood. For example, it is important that the polymer not cause lysing of the blood cells. Furthermore, the charge of the polymer must be adequate to effect maintenance of phase separation, without being so strong as to set up a charge gradient causing ion leakage through the membranes of the blood cells. As an example of how polymers can be satisfactory for sewage separation but not for blood separation, it will be shown hereinafter that some polymers with quaternary amines do not work satisfactorily.
On the other hand, U.S. Pat. No. 3,902,964 describes coagulator accelerators that are used strictly with whole blood to coagulate blood cells in an accelerated manner. However, two such agents are required--Polybrene and a lectin. Furthermore, Polybrene is hexadimethrine bromide, a polymeric structure that does not lend itself to significant structural modification to provide workable alternatives. In contrast, vinyl addition polymers are much more versatile. However, as will become apparent, not any vinyl addition polymer is effective.
There has been a need, therefore, prior to this invention, to identify a class of coagulator accelerators capable of variation, and which will cause permanent coagulation of blood cells during centrifuging, without lysing and without requiring other accelerators as well.